Fröhlich et al. Cancer Cell Int (2016) 16:68 DOI 10.1186/s12935-016-0343-0 Cancer Cell International PRIMARY RESEARCH Open Access Molecular mechanism leading to SAHA‑induced autophagy in tumor cells: evidence for a p53‑dependent pathway Leopold F. Fröhlich1,2*†, Maria Mrakovcic3†, Claudia Smole1 and Kurt Zatloukal1 Abstract Background: Recent studies indicated that histone deacetylase inhibitors (HDACi), a class of anticancer agents, are in addition to their ability of apoptosis induction also capable of provoking autophagy. Promoted by the treatment of malignant uterine sarcoma cells with the HDACi suberoylanilide hydroxamic acid (SAHA), we previously demonstrated predominant dose-dependent activation of autophagy in ESS-1 cells, but prevalent induction of apoptosis in MES-SA cells. Methods: In order to extend our previous studies, SAHA-treated ESS-1 and MES-SA cells were monitored for protein expression to reveal differences in known markers of apoptosis explaining the different cytotoxic responses. Further analysis of the identified candidate protein included cell rescue experiments by gene transfer followed by subsequent screening of cells for induction of apoptosis and autophagy by immunoblotting, caspase activity as well as LC3 and MDC/PI staining. LDH release assays were performed to assess the amount of cell-mediated cytotoxicity. Results: In our search for responsible autophagic regulatory genes upstream of mammalian target of rapamycin (mTOR), we now discovered that, in contrast to MES-SA cells, a TP53-637C>T nonsense mutation located in the trans- activating domain of the oncogenic suppressor p53 causes loss of its protein and consequently reduced PUMA induc- tion in ESS-1 cells. Upon re-introduction of wild-type TP53, SAHA-treated ESS-1 cells underwent immediate apoptotic cell death as supported by upregulation of PUMA and caspase-9 as well as by activation of caspases-3 and -7 and PARP-1 cleavage. Concurrent downregulation of autophagy was noticed by upregulated mTor and phospho-mTOR expression as well as monitoring autophagosome formation employing LC3 and MDC staining. Previously, cytoplas- mic master regulatory activities of the oncogenic suppressor p53 in inhibiting autophagy and triggering apoptosis were unravelled. Accordingly, p53-deficiency could explain both, the previously documented apoptosis resistance and prevailing SAHA-induced autophagy in ESS-1 cells. Using MES-SA cells with RNAi-silenced p53 expression and several p53-deficient tumor cell lines undergoing SAHA-induced autophagy, we could generally validate our finding suggesting an inhibitory role for p53 in the autophagic pathway in response to SAHA treatment. Conclusions: Conclusively, these results could identify cytoplasmic p53 protein as a molecular switch that directly mediates the cytotoxic response of SAHA and thus open new therapeutic avenues. Keywords: HDACi, SAHA, Autophagy, Apoptosis, p53, HDACi, ESS-1, MES-SA, Tumor *Correspondence: [email protected] †Leopold F. Fröhlich and Maria Mrakovcic contributed equally to this work 2 Department of Cranio‑Maxillofacial Surgery, University of Münster, Albert‑Schweitzer‑Campus 1, 48149 Münster, Germany Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fröhlich et al. Cancer Cell Int (2016) 16:68 Page 2 of 15 Background conjugation to ubiquitin-like conjugation systems, cyto- HDACi are a well-characterized class of cancer therapeu- solic LC3 (termed LC3-I) gets incorporated to the grow- tic agents with promising clinical activity against hemato- ing autophagosome structure (then termed LC3-II) and logic and solid tumors at well tolerated doses by patients therefore acts as a marker [16, 17]. [1, 2]. HDAC expression is essential in the establishment Currently, molecular mechanisms by which SAHA of a transcriptionally inactive chromatin structure by modulates and thereby initiates autophagic induction posttranslationally modifying histone and nonhistone upstream of mTOR remain unknown. In the absence acetylation sites [3]. Counteracting the effects of HDACs of any observed transcriptional regulation of mTOR or by inhibition of HDAC activity therefore induces chro- upstream pathway components, the possibility remains matin relaxation which leads to altered expression of that SAHA suppresses mTOR by interfering with the only few but important genes involved in the regulation acetylation of regulatory nonhistone proteins. In this of many disbalanced tumor cell processes as cell cycle regard, the transcription of LC3 itself was found to be arrest, differentiation, and apoptosis. In various tumor upregulated by SAHA, as well as downregulated by p53 cell lines, treatment with HDACi most frequently induces which seems to support cancer cell survival under star- apoptosis by sequential activation of a series of cysteine- vation conditions; nevertheless, LC3 itself is not capable dependent aspartate-directed proteases, called caspases of inducing autophagy [12, 18]. Previously, consistently [4, 5]. Nevertheless, their precise mode of function in detected upregulated expression of the class II enzyme, selectively eliminating malignant cells remains unclear. HDAC2, in endometrial stroma sarcoma (ESS) led us Suberoylanilide hydroxamic acid (SAHA), an inhibi- to study the therapeutic options of the HDACi SAHA tor of class I and II HDACs, was previously admitted for [19]. Our group verified that SAHA exerts cytotoxic- therapeutic treatment of cutaneous T cell lymphoma ity on uterine sarcoma cells and significantly prevented [6]. As a single molecule, SAHA has broad range effects tumor cell proliferation by increasing expression of which include the inhibition of cell proliferation by the cell cycle kinase p21WAF1 and decreasing expres- blocking cell cycle progression, the suppression of angio- sion of HDAC2 and 7 in vitro [10]. The number of cells genesis, the induction of cellular differentiation, as well as was decreased in a time- and dose-dependent manner apoptosis in tumor cells [7, 8]. Among other studies, we whereby an established working dose of 3 µM SAHA sig- previously demonstrated that SAHA can, in addition to nificantly reduced ESS-1 cells by 80 % and MES-SA cells mitochondria-mediated apoptosis, also promote caspase- by 48 % after 24 h of treatment, and inhibited the G1/S independent autophagic cell death [9–11] which offers transition [20]. Moreover, we documented pronounced an advantage in overcoming apoptotic resistant tumor activation of apoptosis in MES-SA uterine sarcoma cells. This coincided with decreased expression of the cells in xenografted tumors and in vitro [21], but pre- autophagic key molecular determinant mTOR, a master dominant SAHA-mediated dose-dependent autophagic regulator of cellular metabolism which is a therapeutic cell death in malignant ESS-1 cells accompanied by target for anticancer treatment. Furthermore, phospho- decreased expression of mTOR [10]. During further S6 ribosomal protein (S6rp) which controls cell cycle experiments with combined SAHA and tumor necrosis progression was found to be downregulated. The studies factor-related apoptosis-inducing ligand (TRAIL) treat- of Gammoh et al. supported SAHA-mediated suppres- ment, we noticed a rapidly enhanced cytotoxic effect sion of mTOR during activation of autophagy and further which led to complete cell death after 24 to 48 h. In both elaborated its downstream pathway [12]. It was found tumor cell lines, induction of combined SAHA- and that the nutrient-sensing kinase prevents the formation of TRAIL-induced apoptosis was accompanied by upregu- autophagy by phosphorylation and thereby inactivation lation of the intrinsic/mitochondrial apoptotic pathway of the ULK1 complex; the important role of this further [20]. In the present study, we wanted to further clarify central component in autophagy was previously demon- the cause for the different mode of cell death in both strated by ULK1-deficient cells. mTOR inactivation by sarcoma cell lines related to single SAHA treatment SAHA thus rescues ULK1 activation and thereby induces and thereby elucidate molecular mechanisms provoking autophagy. ULK1 (and PI3 K) complexes subsequentially SAHA-mediated autophagy. activate the proper autophagy-related proteins including the ATG12-ATG5 and LC3-phosphatidylethanolamine Methods complexes that are directly involved in formation of the Chemicals mature autophagosome [13, 14]. The later involves the SAHA was purchased from Alexis Biochemicals (Lausen, nucleation and elongation of the double-membrane vesi- Switzerland). A 10 mM stock solution was prepared cle and its fusion with the lysosome leading to the final with dimethyl sulfoxide (DMSO) and stored at −20 °C. degradation of the autophagosome content [15]. Upon DMSO never exceeded a concentration of 0006 % in Fröhlich et al. Cancer Cell Int (2016) 16:68 Page 3 of 15 any experiment and therefore did not interfere with cell (GATC Biotech AG; Cologne, Germany).